The invention relates to genetically transformed plants that develop larger storage organs. More specifically this invention relates to plants comprising a heterologous gene encoding an enzyme involved in NAD(P)H consumption. These plants develop larger roots, exhibit increased growth, and increased stress tolerance.
Perennial crops, including many forage crops, persist in cultivated fields for several years. Because these plants are capable of multiple cycles of regrowth and harvest, the growth and development are distinctly different than annual grain crops. In most perennial forage production systems, for example alfalfa (Medicago sativa), plants are defoliated before any seed is produced and, unlike annual crops, regrow new vegetative shoots from crown or axillary buds. The energy, carbon, nitrogen and other reserves necessary to support this regrowth come from the root system and crown. The reserves in the root and crown are depleted as the new shoots develop new leaves, which capture light energy for photosynthesis. At a certain stage of development the shoot becomes self-sufficient and obtains its energy and carbon requirements from photosynthesis. At a later stage, the shoot has excess energy and carbon from photosynthesis and exports the excess to the root and crown system to support nitrogen fixation, nutrient uptake and replenishment of reserves. Replenishment of reserves continues until the plant is defoliated again by either grazing, harvesting or natural stresses, for example, freezing.
Roots or other storage organs of most forage legumes convert the imported sucrose to starch, whereas forage grasses store fructans. The rate of starch or fructan accumulation in these storage organs is usually not controlled by the supply of sucrose from the leaf. Instead, sink strength is determined by the ability of the storage organ to synthesize and store starch or fructans. This is determined in turn by the number of amyloplasts (sites of starch storage) in the cell, the number of storage cells in the root, and the metabolism of the cell.
The size of the root system and the quantity of stored nutrient reserves that are available to support new shoot growth determines the rate and amount of shoot regrowth and the economic yield of perennial plants. The agronomic management of forage crops for example is specifically designed to maximize the quantities of these reserves that may accumulate between harvests (Hanson et al., 1988; Barnes et al., 1995). The quantity of stored nutrient reserves determines the ability of the plant to survive winter. Therefore, in northern climates, crop production recommendations include clear guidelines to avoid harvesting alfalfa and other forage crops during late summer and early autumn because these reserves are being replenished for winter
The performance index of alfalfa for example after several winter stresses has been related to the size of the roots. Significant correlations of root mass were found with performance after flooding and icing stress, and a correlation (not significant at the 5% level) was found with freezing stress (Bowley and McKersie, 1990). Poot size alone appears to have an effect on the performance of alfalfa following winter.
Therefore, there is a need to increase the sink strength of the roots of plants. Increased sink strength would increase the amount of carbohydrate and other stored nutrients in roots or other storage organs. The increased levels of reserves would increase the regrowth rate, yield potential, and the likelihood that a perennial plant would survive winter. Similarly, increased reserve levels would ensure yield potential of an annual under varying environmental conditions.
Genetic transformation has been previously used to modify source-sink relationships in plants. Although attempts to improve photosynthesis and thereby increase the export of sucrose to sink organs have not been successful, the modification of carbohydrate metabolism in the sink organ has increased the size of potato tubers. U.S. Pat. No. 5,436,394 discloses the modification of the distribution of photoassimilates, including sucrose, in transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers. Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant. Several plants exhibited phenotypes comprising reduced internode distances and severly reduced root growth.
U.S. Pat. No. 5,723,757 discloses the use of a patatin promoter to drive the expression of gene of interest within a sink organ, such as a root, within transgenic plants. In U.S. Pat. No. 5,750,869, transgenic plants that ectopically express sucrose phophate synthase are shown to exibit altered sink capacities, with increased levels of sucrose, starch and cellulose observed within the sink tissue. Neither of these documents observed increased root growth, or root size, in the transformed plants. Furthermore, the ability of the transgenic plant to withstand stresses WAS not contemplated.
In order to increase the sink strength and size of roots and other storage organs, it may be necessary to directly stimulate the growth and development of the organ. In WO 98/03631, increased growth of main and lateral roots was noted in Arabidopsis transformed with a nucleic acid encoding mitotic cyclin proteins, preferrably the cyclaAt protein. However, there is no indication that these plants exhibited increased regrowth potential, nor that they had increased stress tolerance.
U.S. Pat. No. 5,554,530 discloses an increased tolerance to salt stress and drought resistance in plants transformed with xcex4-pyrroline-5-carboxylic synthetase. Transformed plants exhibited higher levels of proline and improved root growth under salt stress conditions. However, regrowth potential in pernnial plants was not considered. Other stress tolerant plants have been produced by transforming plants with superoxide dismutase (EP 359,617 and EP 356,061), however, no increase in root growth or root size was observed. The regrowth potential in perennial plants was also not considered.
U.S. Pat. No. 5,821,398 discloses the production of transgenic plants expressing alchohol dehydrogenase (ADH) under the control of a fruit specific, inducible promoter, preferrably the tomato ADH2 promoter. Expression of ADH within fruits results in controlled fruit softening and increased flavor content.
In U.S. Pat. No. 5,855,881, the expression of mammalian ADH within plants to produce a ready source of ADH for use as a dietary supplement to ameliorate the effects of alcohol consumption in an animal is discussed. There is no teaching of producing plants expressing ADH that exhibit the properties of increased stress tolerance or increased regrowth potential.
It is an object of the invention to overcome disadvantages of the prior art. This object is met by the combinations of features of the main claims, the sub claims disclose further advantageous embodiments of the invention.
The present invention is directed to introducing at least one heterologous gene into a plant encoding an enzyme involved in consuming NAD(P)H, for example, but not limited to, alcohol dehydrogenase. These plants have increased storage organ mass, such as roots, and the sink strength of the plant is also increased. Furthermore, these plants exhibit increased stress tolerance, and exhibit increased regrowth potential, within and in perennial plants, between growth seasons.
This invention relates to genetically transformed plants that develop larger storage organs. More specifically this invention relates to plants comprising a heterologous gene encoding an enzyme involved in NAD(P)H consumption. These plants develop larger roots, exhibit increased growth, and increased stress tolerance
According to the present invention there is provided a method of increasing the mass of a storage organ of a plant, comprising:
i) transforming the plant with at least one heterologous gene that encodes at least one enzyme that results in NAD(P)H consumption to produce a transformed plant;
ii) selecting the transformed plant for occurrence of the heterologous gene;
iii) growing the transformed plant.
Preferably, the heterologous gene encodes an enzyme that is directly involved in NAD(P)H consumption, selected from the group consisting of alcohol dehydrogenase, glutathione reductase, dehydroxyascorbate reductase, monodehydroascorbate reductase, mitochondrial alternative oxidase, NADH oxidase, and NADPH oxidase. However, the heterologous gene may also encode an enzyme indirectly involved in NAD(P)H consumption, selected from the group consisting of superoxide dismutase, ascorbate peroxidase, and dehydroxyascorbate reductase.
The present invention also pertains to a method for increasing the mass of a storage organ of a plant, comprising:
i) transforming the plant with two distinct heterologous genes that encode enzymes that results in NAD(P)H consumption to produce a transformed plant;
ii) selecting the transformed plant for occurrence of the heterologous genes;
iii) growing the transformed plant.
This invention also relates to a vector comprising a regulatory element in operative association with at least one heterologous gene, wherein the heterologous gene, when expressed in a plant, encodes an enzyme that consumes NAD(P)H and produces a transformed plant characterized in having increased storage organ mass. Preferably, the regulatory element is active in the storage organ, and is root specific.
Furthermore, this invention also relates to a vector comprising two regulatory elements in operative association with two distinct heterologous genes, wherein expression of the heterologous genes in a plant results in the plant having increased storage organ mass. At least one of the regulatory elements is active in the storage organ, and is root specific.
This invention is directed to a transgenic plant, transgenic plant cell, and transgenic seed comprising the either of the vectors defined above. The transgenic plant may be a perennial or an annual plant. If perennial, the plant is selected from the group consisting of strawberries, raspberries, grapevines, apple, roses, orchard grass, brome grass, timothy, ryegrass, fescue, alfalfa, clover, birdsfoot trefoil, turfgrass, bentgrass and bluegrass. If annual, the plant is a winter annual plant or a root crop selected from the group consisting of Brassica spp., wheat, barley, oats, rye, canola, maize, rice, barely, soybean, potatoes and Phaseolus spp
The present invention embraces a method of increasing the tolerance to an environmental stress of a plant, comprising:
i) transforming the plant with at least one heterologous gene that encodes an enzyme that results in NAD(P)H consumption;
ii) selecting the transformed plant for occurrence of the heterologous gene;
iii) growing the transformed plant.
This method also pertains to increasing flooding, freezing, desication, or drought resistance or a combination thereof.
The invention includes an expression system for increasing the size of the storage organs of a plant or to impart greater sink strength in the storage organs, for example, including but not limited to roots, crowns, rhizomes, stolons, tubers, culmns, basal stems and tap roots. The invention also includes a transgenic plant, a plant part, a seed, a plant cell and a plant tissue that includes the expression system. The invention includes the use of the expression system to increase the size of storage organs and the sink strength of the plant.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.